This project presents a continuation of the previous studies on platelet microtubule protein. There are 4 major objectives to this proposal. First, a detailed investigation of the interaction of microtbules with other structural platelet elements, especially actin, myosin, membranes and subcellular organelles. Second, an investigation of cellular factors involved in stabilization or change of equilibrium between polymerized and depolymerized platelet tubulin. Third, a study of the process by which "stimulation" of platelets modifies the state of polymerization of tubulin. Fourth, delineation of the significance of microtubules for platelet function under experimental conditions which will nullify the stimulant effect on cAMP levels of their disruption by antimitotic agents. All these studies will be carried out with human platelets. The methodology for probing possible interactions between microtubules and other platelet structures will range from viscometry, to cosedimentation, to energy transfer studies between acceptor-donor pairs of fluorophores conjugated to different molecules, to investigation of the products of cross-linking produced by bifunctional agents and analyzed by 2-dimensional electrophoresis. The kinetics of microtubule assembly and disassembly will be followed by turbidimetric and ultracenrifugal methods supplemented by electron microscopy. The dynamic equilibrium between polymerized and depolymerized tubulin as it is influenced by a variety of factors will be studied in intact platelets by stabilizing the respective pool of microtubule protein with a mixture of dimethylsulfoxide-glycerol and quantifying tubulin by its colchicine binding activity. The significance of microtubules for a variety of platelet functions including aggregation, adhesion, phagocytosis and migration, random and directed, will be investigated. These studies are important because marked differences exist with respect to functional and biochemical characteristics between tubulins from different sources. The proposed studies will mean a substantial advance in our knowledge of microtubules in an anucleated cell and may yield practical benefits for platelet preservation.